Discovery of diarylpyrimidine derivatives bearing piperazine sulfonyl as potent HIV-1 nonnucleoside reverse transcriptase inhibitors

HIV-1 reverse transcriptase is one of the most attractive targets for the treatment of AIDS. However, the rapid emergence of drug-resistant strains and unsatisfactory drug-like properties seriously limit the clinical application of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Here we show that a series of piperazine sulfonyl-bearing diarylpyrimidine-based NNRTIs were designed to improve the potency against wild-type and NNRTI-resistant strains by enhancing backbone-binding interactions. Among them, compound 18b1 demonstrates single-digit nanomolar potency against the wild-type and five mutant HIV-1 strains, which is significantly better than the approved drug etravirine. The co-crystal structure analysis and molecular dynamics simulation studies were conducted to explain the broad-spectrum inhibitory activity of 18b1 against reverse transcriptase variants. Besides, compound 18b1 demonstrates improved water solubility, cytochrome P450 liability, and other pharmacokinetic properties compared to the currently approved diarylpyrimidine (DAPY) NNRTIs. Therefore, we consider compound 18b1 a potential lead compound worthy of further study.

A cquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV) 1 . Nearly 38.4 million people worldwide were infected by HIV in 2021, which remains a pandemic health issue [https://www.unaids.org/en (EB/OL)]. HIV-1 reverse transcriptase (RT) has important biochemical functions for viral replication as RNA/DNA-dependent DNA polymerase and ribonuclease H (RNase H). Thus, it has been considered as one of the most attractive targets for the treatment of AIDS 2 . Currently, RT inhibitors are mainly divided into nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) 3 . As allosteric inhibitors, NNRTIs interfere with the normal function of RT by binding to the NNRTI binding pocket (NNIBP) that is located about 10 Å from the polymerase active site 4 . NNRTIs have been key components in highly active antiretroviral therapy (HAART) due to their promising anti-HIV-1 activities, high specificity, and relatively low toxicity 5,6 .
As shown in Fig. 1, Nevirapine (NVP, 1), efavirenz (EFV, 2), etravirine (ETR, 3), rilpivirine (RPV, 4) and doravirine (DOR,5) are NNRTIs that have been approved by the U.S. FDA for the treatment of AIDS 7 . NNRTIs have a relatively low genetic barrier because of their allosteric binding, which leads to rapid emergence of drug-resistant strains during their clinical applications 8 . For example, the first-generation NNRTIs NVP and EFV, have shown dramatically reduced activities against the mutant strains K103N, Y181C and L100I 9,10 . However, the second generation of NNRTIs, including ETR, RPV and DOR, demonstrated promising activities against early NNRTI-resistant mutations. Nevertheless, some new resistant strains have been selected by the second generation of NNRTIs, such as K103N/Y181C and F227L/V106A for ETR, E138K and F227C for RPV, and V106A and F227L for DOR 11,12 . ETR and RPV (diarylpyrimidine, DAPY) suffer from poor solubility (ETR, ≪1 μg/mL at pH 7.0; RPV, 20 ng/mL at pH 7.0), which affects their pharmacokinetic (PK) properties 13 . Nevertheless, RPV and other DAPY compounds such as dapivirine (R147681) compensate for low solubility through formation of hydrophobic aggregates that improve their bioavailability 14 , a phenomenon also observed in other relevant cases 15,16 . In addition, ETR and RPV are inhibitors of cytochrome P450 (CYP) enzymes, which can decrease their effective dose, sometimes necessitating co-formulation with PK enhancers such as ritonavir to compensate 17 . Therefore, there is a pressing need to develop novel NNRTIs with improved anti-HIV-1 activities against resistant mutant strains and improved drug-like properties.
The cocrystal structure of ETR/RT (PDB code: 3MEC, Fig. 2a) indicated that the aminobenzonitrile moiety (right-wing) acted on a rather plastic "groove", namely tolerant region I of the HIV-1 NNIBP 18,19 . The tolerant region I, which is formed from V106, F227, L234, P236 and Y318, is a modifiable chemical space that can accommodate various substituents 20 . Taking ETR as the lead compound, our previous efforts have identified a variety of DAPYtyped NNRTIs by exploiting the tolerant region I [19][20][21][22][23] . Particularly, compound BH-11c (6) revealed remarkable anti-HIV-1 activities against the wild-type (WT) and several single mutant strains, including K103N, E138K and Y181C 21 . In addition, BH-11c demonstrated significantly enhanced water solubility (33.4 μg/mL at pH 7.0) and safety profiles compared to ETR. However, BH-11c did not show satisfactory activities toward F227L/V106A and K103N/ Y181C double mutant HIV-1 strains. Hence, it is worth conducting systematical optimization based on BH-11c to yield novel NNRTIs with improved drug-resistance profiles.
Particularly, the F227L/V106A mutation has gained attention due to its resistance to the current NNRTIs, as well as BH-11c 11,21 . A known effective medicinal chemistry strategy to overcome drug resistance is to establish interactions between the ligand and main chain atom(s) of the surrounding amino acid residues 24,25 . Significantly, there is an extended channel in the tolerant region I, consisting of F227 and P236, that leads to the solvent [26][27][28] . In order to better understand the interactions between BH-11c and the NNIBP of HIV-1 RT (PDB code: 3MEC) 29 , molecular docking studies were performed using the software Surflex-Dock SYBYL-X 2.0 30 , and the results were shown in Fig. 2c by PyMOL (http:// www.pymol.org/). In the presence of the methylene group, there is a bond angle of 114.2°between piperazinyl and phenyl rings in the right wing of BH-11c (Fig. 2b). This resulted in the sulfonyl group of BH-11c being 4-6.7 Å away from the main chains of F227, P236 and L234, which was too far to interact. Based on this analysis, we designed a series of HIV-1 inhibitors and the docking results of representative compound 18a1 are shown in Fig. 2c. We speculated that the removal of the methylene group could shorten these distances (~3 Å, Fig. 2c), which potentially form additional interactions with F227, P236 and L234. A nitrogen atom, cyano (CN) or trifluoromethyl group was introduced at position 3 of the right phenyl ring, with the aim of establishing additional interactions with V106. A cyano or cyanoethylene (CV) group was introduced as a privileged fragment in the left-wing. We postulated that such structural optimizations derived from BH-11c could generate additional interactions (especially backbone-binding interactions) with the residues in tolerant region I.
This research focuses on the design, synthesis, anti-HIV evaluation and preliminary structure-activity relationships (SARs) of DAPY-typed derivatives. Furthermore, the co-crystal structure determination and molecular dynamics (MD) simulation were utilized to investigate interaction modes between a representative compound and the binding pocket. Finally, drug-like properties, including water solubility, cytochrome P450 (CYP) inhibition, and PK properties were investigated in detail.
According to the results in Table 1, the preliminary SARs and structure-cytotoxicity relationships (SCRs) can be depicted as follow: 1. By comparing series 17 and 18, it is obvious that the cyanovinyl group at R 1 position is more preferred than the cyano group for anti-HIV-1 activity. However, cyanovinylcontaining compounds exhibited enhanced cytotoxicity possibly due to the "Michael addition effect". 2. Next, we turned our attention to substituents at position 3 of the right phenyl ring (X in scheme 2). By comparing the corresponding derivatives in a, b and c series, the potency of compounds against WT HIV-1 was in the following order: b The cytotoxicity of corresponding compounds in a, b and c series was as follows: when R 1 is cyano group, 17a series (X = N) ≥ 17c series (X = C-CF 3 ) ≥ 17b series (X = C-CN), except compounds 17a2/17c2 and 17b6/17c6; when R 1 is cyanovinyl group, 18a series (X = N) ≥ 18b series (X = C-CN) ≥ 18c series (X = C-CF 3 ). 3. In the case of the terminal substituents on piperazinyl (R 2 ), the order of potency in 17c series was as follow: 17c1 . The terminal substituents in other series have a similar effect on the antiviral activity. Moreover, it is apparent that compounds with an acryloyl substituent indicated higher cytotoxicity than other sulfonyl substituents, except compound 17c6.
WT HIV-1 RT inhibition assay. In order to confirm the drug target of the synthesized DAPY derivatives, representative compounds were selected to test their inhibitory activity against WT HIV-1 RT. As shown in Table 3, these selected compounds exhibited inhibitory effect on HIV-1 RT with IC 50 values between 0.19 to 0.056 μM, being evidently better than RVP (IC 50 = 0.43 μM) and ETR (IC 50 = 1.35 μM). The results indicated that these compounds target HIV-1 RT, and they could be classified as HIV-1 NNRTIs.
Crystal structure of HIV-1 RT in complex with 18b1. We have determined the crystal structure of HIV-1 wild-type (WT) RT in complex with 18b1 at 2.5 Å resolution (Supplementary Table S1 and Supplementary Fig. S1) to gain a thorough understanding of the previously explained SAR. In general, the structure is very similar to previously reported RT-RPV 37 and RT-25a 3 structures (Fig. 5a, Supplementary Fig. S1). Notably, though, the cyano group of 18b1 displays hydrophobic contacts with the side chains of V106 and F227, and the sulfonyl group forms hydrogen bonds with the main chains of F227 and L234 (Fig. 5b). The comparison with previous structures shows that the positioning of 18b1 in the NNIBP is similar to RPV, except for the piperazine sulfonyl moiety that protrudes into the tolerant region I-as in the RT-25a complex (PDB 6C0N)-provoking the uplift of the loop preceding β9 and the one connecting β10-β11 (Fig. 5c). However, the removal of the linking carbon present in 25a (alongside the previously mentioned contacts of the cyano group) accounts for a substantial shift in the piperazine sulfonyl moiety positioning in comparison to the piperidine-linked benzenesulfonamide of 25a. Fig. S2) indicate that the main interactions are similar for 25a and 18b1. A thorough analysis (using http://www.ebi.ac.uk/thorntonsrv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=index.html) reveals that 18b1 has a larger number of non-bonded contacts, especially in terms of backbone interactions (see log files in the Supplementary Information).

Comparison of the protein-ligand contacts (Supplementary
Molecular dynamics (MD) simulation study. Subsequently, the MD simulation was conducted to further explain the differences in the inhibitory activity of 18b1 and ETR against RT variants. Three x-ray structures of HIV-1 RTs 3 , including K103N RT (PDB code: 6C0O), E138K RT (PDB code: 6C0P) and V106A/F227L RT (PDB code: 6DUF) were downloaded from RCSB PDB (rcsb.org) 38 . Figure 6 shows the root mean square deviation (RMSD) of 18b1 and ETR during 300 ns MD simulation with corresponding RT variants. It is obvious that 18b1 has clustered into distinct conformations, which is clear from the pattern and the range of RMSD values. On the contrary, the RMSD plot for ETR shows a stable binding conformation with protein. The most dominant cluster analysis of 18b1 with corresponding RT variants shows that they have similar binding modes (Fig. 7a, c, e). However, the conformation of piperazinyl and the orientation of methyl sulfone are varied. The cyano group of 18b1 is orientated towards the left wing, except K103N variant, which suggest the unique orientation for the cyano group is caused by the mutation of K103 to N103 when binding this variant. The binding modes of representative structure of the most dominant clusters of 18b1 and ETR bound to the three RT variants are similar regarding the orientation of right and left wings of both compounds in all RT variants binding (Fig. 7). Figure 7 shows the binding poses of 18b1 and ETR to the binding sites of the K103N, E138K and V106A/F227L RT variants in the most abundant cluster of each binding. Supplementary  Table S2 shows detailed interactions, hydrogen bonding and hydrophobic interaction, of both compounds to the three RT variants. The general view of Fig. 7 and Supplementary Table S2 shows that 18b1 forms more interactions with the surrounding amino acids comparing ETR.
Hydrogen bond interactions between 18b1 and ETR and corresponding RT variants were investigated by hydrogen bond analysis implemented in cpptraj (Supplementary Table S2). Supplementary Table S2 shows that the NH group on the right wing of 18b1 and ETR form hydrogen bonds with the backbone oxygen of K103 (N103 in K103N). Interestingly, in contrast to the interactions with other variants, both inhibitors contact the backbone HN of K101 in E138K variant. This could be explained by the lost hydrogen bond between K101 and K138 in E138K, as presented in the other RT variants according to the hydrogen bond analysis. This made K101 orientates to a position that can interact with the inhibitor more frequently than other variants.
The other type of hydrogen bond takes place between C-H and oxygen or nitrogen atom [39][40][41][42] . It is considered an important bonding force in biomolecules, despite it is a weak interaction. Supplementary Table S2 shows that the oxygen atoms of 18b1 sulfone (O1 and O2) are involved in aliphatic hydrogen bonding with residues F227, L234 and P236 at good frequency. In K103N variant there are interactions to P225, F227, and P236 with sulfone oxygen atoms. Also, it establishes conventional hydrogen bonds with N103, which is not present in the binding of ETR. The interactions of sulfone oxygen atoms with surrounding amino acids added extra bonding forces, which were reflected on the  50 : concentration required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytopathicity, as determined using the MTT method (mean ± SD, Used for comparison. The data were obtained from the same laboratory using the same method (Prof.  Table S2). These interactions are rarely found in the binding of ETR. Forming hydrogen bonds with the backbone atoms has advantage over interactions with the side chain of amino acids since the mutation will have minimal effect on the activity of these inhibitors. This could explain the activity of 18b1 against the mutant variants compared to the activity of ETR.
Hydrophobic interactions were also investigated in Supplementary Table S2. It is clear that the hydrophobic interactions are dominant when binding to RT variants. 18b1 shows interactions with P95 in E138K, whereas it is not present in the interaction of ETR to any RT variant. Interactions with E138 are present between 18b1 and K103N. Also, 18b1 interacts with P236 in all variants. These extra hydrophobic interactions and the abovementioned hydrogen bonds suggest 18b1 behaves better potency comparing ETR, which is consistent with its experimental activity.  The binding free energies of 18b1 and ETR with corresponding RT variants were calculated using MMPBSA 43 and were represented in Supplementary Table S3. The Van der Waals (vdw) contribution for 18b1 bound to the variants is higher (more negative E vdw ) than the contribution of ETR, which shows the importance of hydrophobic interactions in their activities. Also, it is clear that the contribution to V106A/F227L is the highest followed by E138K and K103N. The nonpolar contribution to the solvation energy shows favorable binding of both 18b1 and ETR with a clear preference to the binding of 18b1. The electrostatic contribution was the highest for K103N followed by V106A/F227L and E138K. The electrostatic contribution to the solvation free energy was the highest for V106A/F227L, followed by K103N and E138K, which shows effect on the binding of 18b1 to each variant. Supplementary Fig. S3 shows the free energy decomposition of binding site amino acids, which elucidates amino acid binding energies, individually (vdw, electrostatic, polar solvation and nonpolar solvation energies). The electrostatic contribution of E138 favors the binding of both 18b1 and ETR to all variants except E138K. According to Supplementary Fig. S3, vdw contribution of E138 favors the binding of 18b1 in all variants, except in E138K which slightly favors the binding of ETR. The mutation of E138 to K138 made the contribution of the polar solvation energy favored, where it is negative in E138K and positive in other variants. Inspecting the vdw interactions in Supplementary Fig. S3 shows that the majority of amino acids favor the binding of 18b1 to all RT variants. Also, the electrostatic energy of most amino acids favors the binding of 18b1 to all RT variants. Furthermore, the nonpolar salvation energy is more negative in the binding of 18b1 to all RT variants.
Fsp 3 value, water solubility, milogP and ligand efficiency. "Fraction of sp 3 carbon atoms" (Fsp 3 ) is used to characterize the carbon saturation of molecules, and it is considered an important parameter for drug-like properties 44,45 . According to the statistics, about 84% of approved drugs possess Fsp 3 values above 0.42 19,41 . As indicated in Table 4, ETR suffers from a low Fsp 3 value (0.10) due to the presence of multiple aromatic fragments, which led to poor water solubility (<1 μg/mL at pH 7.0) and undesirable milogP (5.03). By introduction of piperazine methylsulfonyl, 18b1 possessed acceptable water solubility (13.46 μg/mL at pH 7.0) and reasonable milogP (4.52) with an improved Fsp 3 content (0.27). Furthermore, ligand efficiency (LE) as an emerging index is used to evaluate the balance between biological activity and druglikeness 46,47 . A promising lead compound is considered to have an LE value above 0.3 48,49 , so 18b1 displayed appropriate LE value (0.32) as with ETR (0.41).
In vitro cytochrome P450 (CYP) enzymatic inhibitory activity. It is reported that ETR and RPV inhibit CYP enzymes, which could cause potential drug-drug interactions and limit the co- administration of multiple drugs 50 . Therefore, 18b1 was assessed for its inhibitory potency against the five main CYP isozymes. ETR and selected inhibitors were used as control. As shown in Table 5, 18b1 exhibited weak inhibitory activity against CYP2C9 (IC 50 = 5.30 μM) and CYP2C19 (IC 50 = 8.00 μM), while conversely ETR (IC 50 = 0.277 μM and 0.496 μM, respectively) and RPV (IC 50 = 0.346 μM and 0.335 μM, respectively) were submicromolar CYP2C inhibitors. On the other hand, 18b1 and ETR demonstrated no obvious inhibitory effect (>10 μM) towards CYP1A2, CYP2D6 and CYP3A4. These results indicate that 18b1 is less likely to cause CYP-mediated drug-drug interactions.
In vivo pharmacokinetics study. Compound 18b1 was further selected to evaluate its pharmacokinetics (PK) profile in Sprague-Dawley (SD) rat model. As described in Table 6, 18b1 demonstrated acceptable terminal half-life (T 1/2 = 2.42 h), moderate clearance (CL = 2.54 L/h/kg) and favorable distribution volume (V = 8.81 L/kg) after an intravenous dose of 2.0 mg/kg. When orally administered at a dose of 10.0 mg/kg, the maximum concentration (T max ) of 18b1 was 16.05 ng/mL at 3.50 h (T max ) and the T 1/2 was 3.05 h. Nevertheless, the oral bioavailability (F) of 18b1 was detected to be 1.34%, which requires further optimization, though this is more serious for ETR (undetectable 51 ).

Discussion
In this research, compound BH-11c was utilized as a lead from which we designed a series of DAPY-typed NNRTIs, aiming at enhancing backbone-binding interactions with the residues in tolerant region I. Interestingly, the newly synthesized compound 18b1 demonstrated significantly improved antiretroviral activities compared to BH-11c against all the tested HIV-1 strains. Furthermore, 18b1 possessed single-digit nanomolar potency against the wild-type and five mutant HIV-1 strains, including L100I, K103N, Y181C, E138K and F227L/V106A. The co-crystal structure indicated that the sulfonyl group of 18b1 formed hydrogen bonds with main-chain atoms of F227 and L234, and the cyano group displayed hydrophobic contacts with side-chain atoms of V106 and F227. Further MD simulation studies were conducted   3 hybridized carbons/total carbon count. b Measured with HPLC method (mean ± SD, n = 2). c miLog P = molinspiration predicted Log P. d LE = calculated by the formula − ΔG/HA (non-hydrogen atom), in which normalizing binding energy ΔG = − RT ln Kd, presuming Kd ≈ EC50 (IIIB); R = 1.987 × 10 −3 kcal K −1 mol −1 , T = 298 K. e See ref. 21 .
to explain the differences in the inhibitory activity of 18b1 and ETR against RT variants. Compared to ETR (<1 μg/mL at pH 7.0), 18b1 displayed improved water solubility (13.46 μg/mL at pH 7.0) with an appropriate LE value (0.32). Moreover, 18b1 revealed significantly lower inhibitory activity than ETR and RPV against CYP2C9 and CYP2C19, indicating that 18b1 was less likely to cause drug-drug interactions. Nevertheless, 18b1 requires further optimization to improve the oral bioavailability (F = 1.34%). Consequently, we consider 18b1 as a promising lead compound worthy of further study.